A series of 6-substituted carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators were discovered through 6-position modification guided by insights from the crystallographic profiles of the “short” inverse agonist 6. With the increase in the size of the 6-position substituents, the “short” inverse agonist 6 first reversed its function to agonists and then to “long” inverse agonists. The cocrystal structures of RORγt complexed with the representative “short” inverse agonist 6 (PDB: 6LOB), the agonist 7d (PDB: 6LOA) and the “long” inverse agonist 7h (PDB: 6LO9) were revealed by X-ray analysis. However, minor differences were found in the binding modes of “short” inverse agonist 6 and “long” inverse agonist 7h. To further reveal the molecular mechanisms of different RORγt inverse agonists, we performed molecular dynamics simulations and found that “short” or “long” inverse agonists led to different behaviors of helixes H11, H11’, and H12 of RORγt. The “short” inverse agonist 6 destabilizes H11’ and dislocates H12, while the “long” inverse agonist 7h separates H11 and unwinds H12. The results indicate that the two types of inverse agonists may behave differently in downstream signaling, which may help identify novel inverse agonists with different regulatory mechanisms.
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We are extremely grateful to the National Centre for Protein Science Shanghai (Protein Expression and Purification system) for their instrument support and technical assistance. We thank the staff from the BL17U1 and BL19U1 beamlines at the Shanghai Synchrotron Radiation Facility (SSRF) for assistance during data collection. We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Grant Numbers: 81703415; 81573276; 81874287; 81973163), the K. C. Wong Education to CL, the Fudan-SIMM Joint Research Fund (Grant Number: FUSIMM20174007), the CAS Strategic Priority Research Program (Grant Number: XDA12020372), the Shanghai Biopharmaceutical Science and Technology Supporting Plan (Grant Number: 17431902100; 19431900100), the National Science and Technology Major Project (Grant Number: 2018ZX09711002-003-014), the Natural Science Foundation of Shanghai (Grant Number: 19ZR1436700), and the China Postdoctoral Science Foundation (Grant Number: 2019M651383).
The authors declare no competing interests.
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Yu, Mc., Yang, F., Ding, Xy. et al. Crystallography-guided discovery of carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators: insights into different protein behaviors with “short” and “long” inverse agonists. Acta Pharmacol Sin (2020). https://doi.org/10.1038/s41401-020-00552-w
- cocrystal structures
- MD simulation
- “short” inverse agonists
- “long” inverse agonists